Abstract
Exposure of mammalian cells to UV radiation alters gene expression and cell cycle progression; some of these responses may ensure survival or serve as mutation-avoidance mechanisms, lessening the consequences of UV-induced DNA damage. We showed previously that UV irradiation increases phosphorylation of the p34 subunit of human replication protein A (RPA) and that this hyperphosphorylation correlated with loss of activity of the DNA replication complex. To characterize further the role of RPA hyperphosphorylation in the cellular response to UV irradiation and to determine which protein kinases might be involved, we identified by phosphopeptide analysis the sites phosphorylated in the p34 subunit of RPA (RPA-p34) from HeLa cells before and after exposure to 30 J/m2 UV light. In unirradiated HeLa cells, RPA-p34 is phosphorylated primarily at Ser-23 and Ser-29. At least four of the eight serines and one threonine in the N-terminal 33 residues of RPA-p34 can become phosphorylated after UV irradiation. Two of these sites (Ser-23 and Ser-29) are known to be sites phosphorylated by Cdc2 kinase; two others (Thr-21 and Ser-33) are consensus sites for the DNA-dependent protein kinase (DNA-PK); the fifth site (Ser-11, -12, or -13) does not correspond to the (Ser/Thr)-Gln DNA-PK consensus. All five can be phosphorylated in vitro by incubating purified RPA with purified DNA-PK. Two additional sites, probably Ser-4 and Ser-8, are phosphorylated in vivo after UV irradiation and in vitro by purified DNA-PK. The capacity of purified DNA-PK to phosphorylate many of these same sites on RPA-p34 in vitro implicates DNA-PK or a kinase with similar specificity in the UV-induced hyperphosphorylation of RPA in vivo.
Highlights
¶ Present address: Procter & Gamble Far East Inc., Kobe Technical Center, 17 Koyo-cho Naka 1-chome, Higashinada-ku, Kobe 658 Japan
Previous studies established that replication protein A (RPA)-p34 is phosphorylated in a cell cycle-dependent manner in both yeast and mammalian cells [28, 49] beginning at the transition of G1 to S phase and continuing until mitosis
Consistent with these in vivo studies, it was demonstrated that RPA-p34 is phosphorylated in vitro during SV40 T antigen-dependent DNA replication in a cell-free system [30, 50]
Summary
¶ Present address: Procter & Gamble Far East Inc., Kobe Technical Center, 17 Koyo-cho Naka 1-chome, Higashinada-ku, Kobe 658 Japan. We and others [30, 31] previously showed that RPA-p34 is hyperphosphorylated upon exposure of cultured human cells to UV or ionizing radiation This DNA damageinduced hyperphosphorylation is coincident with cell cycle arrest and loss of the ability of cell extracts to support DNA replication [30]. These observations suggest that phosphorylation of RPA-34 serves as an essential mechanism for modulating RPA activity and its interactions with other proteins. To understand the role of RPA phosphorylation in DNA replication, repair, and recombination, it is necessary to define characteristic phosphorylation sites in specific forms of RPA and to identify the enzyme activities responsible for their creation. In mouse scid cells, which are mutated in the gene for the catalytic subunit of DNA-PK (DNA-PKCS), diminished levels of hyperphosphorylation of RPA were observed after DNA damage, pointing to a role for DNA-PKCS in DNA damage-induced hyperphosphorylation of RPA-p34 [20]
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